The present invention relates to the field of endotoxin analysis.
Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and its activity is mainly attributed to LPS (lipopolysaccharide). In the living body, endotoxin exists as a part of the outer membrane in the surface layer of gram negative bacteria. Generally, after death of gram-negative bacteria, endotoxin is liberated and is present in a free form in blood.
When more than a certain level of endotoxin is present in blood, the endotoxin stimulates monocytes, granulocytes, etc., resulting in excessive production of inflammatory cytokines. Consequently, so called endotoxinemia accompanied by symptoms such as fever, sepsis, septic shock, multiple organ failure, etc. is induced. For this reason, detection of endotoxin in pharmaceuticals for injection, etc. is crucial, and thus the bacterial endotoxin test is prescribed by the Japanese, U.S., and European pharmacopeias. From the aspect of clinical diagnosis, precise measurement of blood endotoxin level is considered crucial for early diagnosis and therapeutic effect evaluation.
Examples of a conventional method for measuring endotoxin include the pyrogen test, in which a rabbit is treated with a direct injection of a test sample and measured for increase in body temperature that can be converted into the endotoxin level, and the Limulus test utilizing gelation of horseshoe crab amebocyte lysate triggered by endotoxin. The method involving direct injection into a rabbit has problems in cost, length of time required to obtain the test results, and sensitivity, and for this reason, the Limulus test currently prevails as a method for measuring endotoxin.
A gelation of horseshoe crab amebocyte lysate is triggered by endotoxin. The gelation process of horseshoe crab amebocyte lysate contains a Factor-C pathway specifically associated with endotoxin. The Factor-C pathway is constituted by the following cascades. First, endotoxin firmly binds with Factor-C, and thereby activates the Factor-C. Then, Factor-C activated by binding with endotoxin (active Factor-C) activates Factor B. Subsequently, activated Factor B (active Factor B) activates a proclotting enzyme, resulting in production of a clotting enzyme. This clotting enzyme partially hydrolyzes its substrate, i.e., coagulogen. As a result, peptide C is liberated from the coagulogen, and a clotting protein, coagulin, is produced. By a coagulation action of the coagulin, gelation occurs.
The Limulus test for measuring endotoxin utilizes the above-mentioned gelation process of horseshoe crab amebocyte lysate triggered by endotoxin. As the Limulus test, a gel-clot technique, a colorimetric technique using synthetic chromogenic substrates, and a kinetic turbidimetric techniques are established techniques used to evaluate gelation (i.e., clotting, see FIG. 1).
New methods of detection are needed that can (i) increase the limit of detection of endotoxin, (ii) reduce the about of amebocyte lysate required for performing the assay, and/or (iii) allow for the monitoring of samples containing light scattering compositions.